EP1313861A2 - 50566 : un nouveau facteur apparente a la glyoxalase ii humaine et utilisations de ce facteur - Google Patents

50566 : un nouveau facteur apparente a la glyoxalase ii humaine et utilisations de ce facteur

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Publication number
EP1313861A2
EP1313861A2 EP01964528A EP01964528A EP1313861A2 EP 1313861 A2 EP1313861 A2 EP 1313861A2 EP 01964528 A EP01964528 A EP 01964528A EP 01964528 A EP01964528 A EP 01964528A EP 1313861 A2 EP1313861 A2 EP 1313861A2
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European Patent Office
Prior art keywords
g2rf
polypeptide
nucleic acid
seq
acid molecule
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EP01964528A
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German (de)
English (en)
Inventor
Rachel Meyers
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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Publication of EP1313861A2 publication Critical patent/EP1313861A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 2-oxaloaldehydes are potentially toxic compounds which can arise through normally functioning metabolic pathways and which must be maintained in proper equilibrium to avoid cellular damage.
  • a glyoxalase system that catalyzes the conversion of 2- oxaloaldehydes into 2-hydrocarboxylic acids using glutathione (GSH) as a co-enzyme.
  • the glyoxalase system comprises two distinct enzymes, glyoxalase I (EC 4.4.1.5 lactoylglutathione lyase) and glyoxalase II (EC 3.1.2.6 hydroxyacylglutathione hydrolase), homologs of which are known to exist in virtually all prokaryotic and eukaryotic organisms (Bito et al. (1991) J. Biol. Chem. 272:21509-21519; Ridderstom et al. (1996) J. Biol. Chem. 271:319-3123).
  • the glyoxalase reactions initiate when GSH reacts non-enzymatically with a 2-oxaloaldehyde, yielding a thiohemiacetyl compound (Reaction A below; see, e.g., Cameron et al. (1999) Structure 7:1067-1078).
  • Glyoxalase I catalyzes the isomerization of the thiohemiacetyl to produce a 2- hydroxy carboxy lie acid (Reaction B below).
  • Glyoxalase II catalyzes the hydrolysis of the thioester to produce GSH and a 2-hydrocarboxylic acid (Reaction C below).
  • methylglyoxal H 3 C-(CO)-CHO
  • Methylgyloxal is formed in higher eukaryotes primarily from the normal glycolytic conversion of triose- phosphates by the enzyme triose-phosphate isomerase (Richard (1993) Biochem. Soc. Trans. 21 :549-553).
  • methylglyoxal arises mainly from the metabolism of dihydroxyacetone phosphate by methylglyoxal synthase (Bito et al. (1991) J. Biol. Chem. 272:21509-21519).
  • Methylglyoxal can produce covalent DNA adducts and will react with arginine and lysine residues in proteins. While this property may potentially play a biological role, it is clear that high levels of methylglyoxal and other oxaloaldehydes are cytotoxic and, thus, must be maintained at controlled levels (Abordo et al. (999) Biochem. Pharmacol. 58:641-648). Accordingly, the detoxification activity of the glyoxalase system has been implicated in a wide array of general cellular functions including proliferation, differentiation, and cell division. For example, a recent report indicates that glyoxalase II plays a role in the regulation of spermatogenesis (Ji et al.
  • the glyoxalase system has also been the focus of some oncological research (Hooper et al. (1988) Cell Mol. Biol. 34:399- 405; Liotti et al. (1993) Bull. Cancer 80:62-68; Thornalley (1995) Crit Rev Oncol Hematol. 20:99-128; Murthy et al. (1994) J Med. Chem. 37:2161-2166). Linkage of the glyoxalase system has also been established for other disorders including diabetes (McLellan et al. (1993) Biochem. Soc. Trans. 21 :172S; Beisswenger et al.
  • the present invention is based, at least in part, on the discovery of .novel family members of the glyoxalase enzyme system, referred to herein as "Glyoxalase II Related Factor” or "G2RF" nucleic acid and polypeptide molecules.
  • G2RF nucleic acid and polypeptide molecules of the present invention are useful as modulating agents in regulating a variety of cellular and/or biological processes, e.g., detoxification, maintenance of metabolite equilibrium, cellular proliferation, tissue differentiation, control of cell cycle, and immune response.
  • this invention provides isolated nucleic acid molecules encoding G2RF polypeptides or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of G2RF-encoding nucleic acids.
  • the invention features an isolated nucleic acid molecule that includes the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO:3.
  • the invention features an isolated nucleic acid molecule that encodes a polypeptide including the amino acid sequence set forth in SEQ ID NO:2.
  • the invention features an isolated nucleic acid molecule that includes the nucleotide sequence contained in the plasmid deposited with ATCC® as Accession
  • the invention features isolated nucleic acid molecules including nucleotide sequences that are substantially identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical) to the nucleotide sequence set forth as SEQ ID NO:l or SEQ ID NO:3.
  • the invention further features isolated nucleic acid molecules including at least 428, 450, 500, 521, 550, 600, 650, 700 or more contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NO:l or SEQ ID NO:3.
  • the invention features isolated nucleic acid molecules which encode a polypeptide including an amino acid sequence that is substantially identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%o identical) to the amino acid sequence set forth as SEQ ID NO:2.
  • the present invention also features nucleic acid molecules which encode allelic variants of the polypeptide having the amino acid sequence set forth as SEQ ID NO:2.
  • the present invention also features nucleic acid molecules which encode fragments, for example, biologically active or antigenic fragments, of the full-length polypeptides of the present invention (e.g., fragments including at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2).
  • the invention features nucleic acid molecules that are complementary to, antisense to, or hybridize under stringent conditions to the isolated nucleic acid molecules described herein.
  • the invention provides vectors including the isolated nucleic acid molecules described herein (e.g., G2RF -encoding nucleic acid molecules). Such vectors can optionally include nucleotide sequences encoding heterologous polypeptides. Also featured are host cells including such vectors (e.g., host cells including vectors suitable for producing G2RF nucleic acid molecules and polypeptides). In another aspect, the invention features isolated G2RF polypeptides and/or biologically active or antigenic fragments thereof.
  • Exemplary embodiments feature a polypeptide including the amino acid sequence set forth as SEQ ID NO:2, a polypeptide including an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 91%, 98% or 99% identical to the amino acid sequence set forth as SEQ ID NO:2, a polypeptide encoded by a nucleic acid molecule including a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth as SEQ ID NO:l or SEQ ID NO:3.
  • fragments of the full-length polypeptides described herein e.g., fragments including at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more contiguous amino acid residues of the sequence set forth as SEQ ID NO:2) as well as allelic variants of the polypeptide having the amino acid sequence set forth as SEQ ID NO:2.
  • the G2RF polypeptides and/or biologically active or antigenic fragments thereof are useful, for example, as reagents or targets in assays applicable to treatment and/or diagnosis of G2RF mediated or related disorders.
  • a G2RF polypeptide or fragment thereof has a G2RF activity.
  • a G2RF polypeptide or fragment thereof has a transmembrane domain, a metallo-beta lactamase superfamily domain, and, optionally, has a G2RF activity.
  • the invention features antibodies (e.g., antibodies which specifically bind to any one of the polypeptides described herein) as well as fusion polypeptides including all or a fragment of a polypeptide described herein.
  • the present invention further features methods for detecting G2RF polypeptides and/or G2RF nucleic acid molecules, such methods featuring, for example, a probe, primer or antibody described herein. Also featured are kits for the detection of G2RF polypeptides and/or G2RF nucleic acid molecules. In a related aspect, the invention features methods for identifying compounds which bind to and/or modulate the activity of a G2RF polypeptide or G2RF nucleic acid molecule described herein. Further featured are methods for modulating a G2RF activity.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human G2RF.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1154 of SEQ ID NO:l .
  • the amino acid sequence corresponds to amino acids 1 to 282 of SEQ ID NO: 2.
  • the coding region without the 5' and 3' untranslated regions of the human G2RF gene is shown in SEQ ID NO: 3.
  • Figure 2 depicts a structural, hydrophobicity, and antigenicity analysis of the human G2RF polypeptide.
  • Figure 3 depicts the results of a search which was performed against the HMM database in PFAM and which resulted in the identification of one "metallo-beta lactamase superfamily domain" in the human G2RF polypeptide (SEQ ID NO:2) ⁇
  • Figure 4 depicts the results of tissue expression analysis of G2RF mRNA using Taqman analysis.
  • the present invention is based, at least in part, on the discovery of novel molecules, referred to herein as "Glyoxalase II Related Factor " or "G2RF" nucleic acid and polypeptide molecules, which are novel members of the glyoxalase system enzyme family.
  • G2RF Glyoxalase II Related Factor
  • These novel molecules are capable of metabolizing toxic compounds (e.g., cytotoxin or other metabolites) in a cell, e.g., a heart, placenta, lung, liver, skeletal muscle, thymus, kidney, pancreas, testis, ovary, prostate, colon, or brain cell. By doing so, these molecules help maintain a proper equilibrium of toxic compounds in a cell, thus preventing the occurrence of cellular damage.
  • toxic compounds e.g., cytotoxin or other metabolites
  • a "glyoxalase II related factor” includes a protein or polypeptide which is involved in the metabolism of cytotoxins and other metabolites, as well as in the regulation of their cellular levels.
  • cytotoxins and “metabolites” include compounds which can be harmful or detrimental to a cell when present in sufficient concentrations or quantities. Cytotoxins and metabolites include those which arise from endogenous sources, e.g., the normal metabolic processes of the cell such as the energetic metabolic pathways. Cytotoxins and metabolites may also enter the cell from the extracellular milieu. Cytotoxins and metabolites which enter the cell include those which originate from outside the organism (xenobiotic compounds).
  • cytotoxins and metabolites include oxaloaldehydes, hydrocarboxylic acids, pharmacological compounds (e.g., chemotherapeutic compounds and anti-cancer drugs), oxidative compounds, glutathione-conjugates, energy metabolites, methylglyoxal, and the like.
  • pharmacological compounds e.g., chemotherapeutic compounds and anti-cancer drugs
  • oxidative compounds e.g., glutathione-conjugates
  • energy metabolites methylglyoxal, and the like.
  • the phrase "regulation of cellular levels” includes cellular mechanisms involved in regulating and influencing the levels (e.g. , intracellular and/or extracellular levels) of cytotoxins and metabolites (e.g., oxaloaldehydes and hydrocarboxylic acids or glutathione-conjugates).
  • cytotoxins and metabolites e.g., oxaloaldehydes and hydrocarboxylic acids or glutathione-conjugates.
  • Such mechanisms include the conversion of potentially cytotoxic compounds into non-toxic or less toxic compounds, e.g., conversion of oxaloaldehydes (such as methylglyoxal or glutathione conjugates) into hydrocarboxylic acids (such as lactate) in response to biological cues, such as formation of nucleotide adjunct, modification of amino acids, and oxidative stress.
  • the G2RF molecules by participating in the regulation of cytotoxin and metabolite levels, may provide novel diagnostic targets and therapeutic agents for controlling cytotoxin- and metabolite-associated disorders (e.g., glyoxalase-associated disorders, oxaloaldehyde- and methylglyoxal-associated disorders).
  • cytotoxin- and metabolite-associated disorders e.g., glyoxalase-associated disorders, oxaloaldehyde- and methylglyoxal-associated disorders.
  • cytotoxin-associated disorders and “metabolite- associated disorders” include disorders, diseases, or conditions which are characterized by aberrant, e.g., upregulated, do nregulated, or misregulated, cytotoxin and/or metabolite levels (e.g., oxaloacetate, hydroxycarboxylic acid, thioester compound, or glutathione-conjugated compound levels).
  • aberrant e.g., upregulated, do nregulated, or misregulated
  • cytotoxin and/or metabolite levels e.g., oxaloacetate, hydroxycarboxylic acid, thioester compound, or glutathione-conjugated compound levels.
  • cardiovascular disorders e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, or arrhythmia.
  • cardiovascular disorders e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary
  • cytotoxin- and metabolite-associated disorders include disorders of the central nervous system, e.g., cystic fibrosis, type 1 neurofibromatosis, cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Creutzfeldt- Jakob disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff s psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive
  • cytotoxin- and metabolite-associated disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety. Still other examples of cytotoxin- and metabolite-associated disorders include cellular proliferation, growth, differentiation, or migration disorders. Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes.
  • a "cellular proliferation, growth, differentiation, or migration process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells (e.g., spermatogenesis), or by which a cell moves closer to or further from a particular location or stimulus.
  • disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.
  • cytotoxin- and metabolite-associated disorders include disorders of the immune system, such as the immune response during starvation, Wiskott-Aldrich syndrome, viral infection, autoimmune disorders or immune deficiency disorders, e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency.
  • Other examples of cytotoxin- and metabolite-associated disorders include congenital malformities, including facio-genital dysplasia; and skin disorders, including microphthalmia with linear skin defects syndrome.
  • family when referring to the polypeptide and nucleic acid molecules of the invention is intended to mean two or more polypeptides or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first polypeptide of human origin, as well as other, distinct polypeptides of human origin or alternatively, can contain homologues of non-human origin, e.g., mouse or monkey polypeptides.
  • Members of a family may also have common functional characteristics.
  • the family of G2RF polypeptides comprise at least one
  • transmembrane domain includes an amino acid sequence of about 20-45 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha- helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, alanines, valines, phenylalanines, prolines or methionines.
  • Transmembrane domains are described in, for example, Zaeaux W.N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference.
  • Amino acid residues 129-145 of the human G2RF polypeptide (SEQ ID NO:2) comprise a transmembrane domain ( Figure 2). Accordingly, G2RF polypeptides having at least 50-60% homology, preferably about 60-70%, more preferably about 10-80%, or about 80-90%) homology with a transmembrane domain of human G2RF are within the scope of the invention.
  • the amino acid sequence of the protein may be subjected to MEMS AT analysis.
  • a MEMSAT analysis resulting in the identification of a transmembrane domain in the amino acid sequence of human G2RF (SEQ ID NO:2) at about residues 129-145 are set forth in Figure 2.
  • a G2RF molecule of the present invention is identified based on the presence of at least one "metallo-beta-lactamase superfamily domain", also referred to interchangeably herein as a "lactamase-B domain.”
  • metalo-beta-lactamase superfamily domain or "lactamase-B domain” includes a protein domain having an amino acid sequence of about 80-250 amino acid residues and has a bit score of at least 80 when compared against a metallo-beta-lactamase superfamily domain Hidden Markov Model (HMM).
  • HMM Hidden Markov Model
  • a "metallo-beta- lactamase superfamily domain” has an amino acid sequence of about 90-240, 100-220, 120-200, 140-180, or more preferably, about 165 amino acid residues, and a bit score of at least 90, 100, 110, 120, or more preferably about 133.3.
  • a "metallo-beta-lactamase superfamily domain” includes a domain which has an amino acid sequence of about 80-250 amino acid residues, and serves to catalyze the hydrolysis of a thioester (e.g. the thioester in a lactoylglutathione compound).
  • Metallo-beta lactamase superfamily domains are described in, for example, Carfi et al., (1995) EMBO Journal 14:4914-4921, the contents of which are incorporated herein by reference.
  • the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database).
  • the metallo-beta-lactamase superfamily domain has been assigned the PFAM Accession No.
  • the G2RF molecules of the invention include at least one transmembrane domain and/or at least one a metallo-beta-lactamase superfamily domain.
  • Isolated G2DF polypeptides of the present invention have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO: 2 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:l or 3.
  • the term "sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains having at least 50%, 55%), 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical.
  • amino acid or nucleotide sequences which share at least 50%, 52%, 53%, 54%), 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity and share a common functional activity are defined herein as sufficiently identical.
  • a G2RF polypeptide includes at least one or more of the following domains: a transmembrane domain and/or a metallo-beta-lactamase superfamily domain, and has an amino acid sequence at least about 50%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • a G2RF polypeptide includes at least one or more of the following domains: a transmembrane domain and/or a metallo- beta-lactamase superfamily domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3.
  • a G2RF polypeptide includes at least one or more of the following domains: a transmembrane domain and/or a metallo-beta-lactamase superfamily domain, and has a G2RF activity.
  • G2RF activity includes an activity exerted by a G2RF polypeptide or nucleic acid molecule on a G2RF responsive cell or tissue, or on a G2RF polypeptide substrate, as determined in vivo, or in vitro, according to standard techniques.
  • a G2RF activity is a direct activity, such as an association with a G2RF- target molecule.
  • a "target molecule” or “binding partner” is a molecule with which a G2RF polypeptide binds or interacts in nature, such that G2RF-mediated function is achieved.
  • a G2RF target molecule can be a non-G2RF molecule, for example, a non-G2RF polypeptide.
  • a G2RF target molecule is a G2RF ligand, e.g., a cytotoxin, a metabolite, glutathione, a gluathione- conjugated compound such as lactoylglutathione, or a thioester-containing compound.
  • a G2RF target molecule can have one or more of the following activities: (1) it may interact with cytotoxins and metabolites (e.g., lactoylglutathione, a glutathione-conjugated metabolite, a hydroxycarboxylic acid, and the like), (2) it may catalyze the mebobolism of a cytotoxin or metabolite (e.g.
  • a G2RF activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the G2RF polypeptide with a G2RF ligand. The biological activities of G2RF are described herein.
  • the G2RF polypeptides of the present invention can have one or more of the following activities: (1) modulation of signal transduction in a cell, (2) modulation of cytotoxin and/or metabolite levels (e.g., detoxification), (3) maintenance of equilibrium of cytotoxins and/or metabolites, (4) modulation of cancer or tumor progression, (5) modulation of cellular proliferation, (6) modulation of tissue development (e.g. embryogenesis), (7) modulation of differentiation, (8) modulation of apoptosis, and (9) modulation of energy metabolism.
  • modulation of signal transduction in a cell modulation of cytotoxin and/or metabolite levels (e.g., detoxification), (3) maintenance of equilibrium of cytotoxins and/or metabolites, (4) modulation of cancer or tumor progression, (5) modulation of cellular proliferation, (6) modulation of tissue development (e.g. embryogenesis), (7) modulation of differentiation, (8) modulation of apoptosis, and (9) modulation of energy metabolism.
  • the nucleotide sequence of the isolated human G2RF cDNA and the predicted amino acid sequence of the human G2RF polypeptide are shown in Figure 1 and in SEQ ID NOs:l and 2, respectively.
  • a plasmid containing the nucleotide sequence encoding human G2RF was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on and assigned Accession
  • the human G2RF gene which is approximately 1154 nucleotides in length, encodes a polypeptide which is approximately 282 amino acid residues in length.
  • nucleic acid molecules that encode G2RF polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify G2RF-encoding nucleic acid molecules (e.g., G2RF mRNA) and fragments for use as PCR primers for the amplification or mutation of G2RF nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated G2RF nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 3, or the nucleotide sequence of the
  • DNA insert of the plasmid deposited with ATCC as Accession Number can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO: 1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , as a hybridization probe,
  • G2RF nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • PCR polymerase chain reaction
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to G2RF nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1.
  • the sequence of SEQ ID NO: 1 corresponds to the human G2RF cDNA.
  • This cDNA comprises sequences encoding the human G2RF polypeptide (i.e., "the coding region", from nucleotides 22-870) as well as 5' untranslated sequences (nucleotides 1-21) and 3' untranslated sequences (nucleotides 871-1154).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO: 1 (e.g. , nucleotides 22-870, corresponding to SEQ ID NO:3).
  • the isolated nucleic acid molecule comprises SEQ ID NO:3 and nucleotides 1-21 and 871-1154 of SEQ ID NO:l.
  • the nucleic acid molecule consists of the nucleotide sequence set forth as SEQ ID NO:l or SEQ ID NO:3.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences.
  • DNA insert of the plasmid deposited with ATCC as Accession Number is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 53%), 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence shown in SEQ ID NO:l or 3 (e.g., to the entire length of the nucleotide sequence), or to the nucleotide sequence (e.g., the entire length of the nucleotide sequence) of the DNA insert of the plasmid deposited with ATCC as
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least (or no greater than) 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000- 1100, or more nucleotides in length and hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a G2RF polypeptide, e.g., a biologically active portion of a G2RF polypeptide.
  • the nucleotide sequence determined from the cloning of the G2RF gene allows for the generation of probes and primers designed for use in identifying and/or cloning other G2RF family members, as well as G2RF homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the probe/primer e.g., oligonucleotide
  • Exemplary probes or primers are at least (or no greater than) 12 or 15, 20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides in length and/or comprise consecutive nucleotides of an isolated nucleic acid molecule described herein. Also included within the scope of the present invention are probes or primers comprising contiguous or consecutive nucleotides of an isolated nucleic acid molecule described herein, but for the difference of 1, 2, 3, 4, 5, 6, 1, 8, 9 or 10 bases within the probe or primer sequence. Probes based on the G2RF nucleotide sequences can be used to detect (e.g., specifically detect) transcripts or genomic sequences encoding the same or homologous polypeptides.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • a set of primers is provided, e.g. , primers suitable for use in a PCR, which can be used to amplify a selected region of a G2RF sequence, e.g., a domain, region, site or other sequence described herein.
  • the primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length.
  • the primers should be identical, or differs by no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases when compared to a sequence disclosed herein or to the sequence of a naturally occurring variant.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a G2RF polypeptide, such as by measuring a level of a G2RF-encoding nucleic acid in a sample of cells from a subject e.g., detecting G2RF mRNA levels or determining whether a genomic G2RF gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a G2RF polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , which encodes a polypeptide having a G2RF biological activity (the biological activities of the G2RF polypeptides are described herein), expressing the encoded portion of the G2RF polypeptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the G2RF polypeptide.
  • the nucleic acid molecule is at least 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, or more nucleotides in length and encodes a polypeptide having a G2RF activity (as described herein).
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number . Such differences can be due to due to degeneracy of the genetic code, thus resulting in a nucleic acid which encodes the same G2RF polypeptides as those encoded by the nucleotide sequence shown in SEQ ID NO:l or 3, or the nucleotide sequence of the
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a polypeptide having an amino acid sequence which differs by at least 1, but no greater than 5, 10, 20, 50 or 100 amino acid residues from the amino acid sequence shown in SEQ ID NO:2, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number .
  • the nucleic acid molecule encodes the amino acid sequence of human G2RF. If an alignment is needed for this comparison, the sequences should be aligned for maximum homology.
  • Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologues (different locus), and orthologues (different organism) or can be non- naturally occurring.
  • Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).
  • Allelic variants result, for example, from DNA sequence polymorphisms within a population (e.g., the human population) that lead to changes in the amino acid sequences of the G2RF polypeptides. Such genetic polymorphisms in the G2RF genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a G2RF polypeptide, preferably a mammalian G2RF polypeptide, and can further include non-coding regulatory sequences, and introns.
  • the invention features isolated nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number , wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:l or SEQ ID NO:3, for example, under stringent hybridization conditions.
  • Allelic variants of human G2RF include both functional and non-functional G2RF polypeptides.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the human G2RF polypeptide that maintain the ability to bind a G2RF ligand or substrate and/or modulate hydrolysis and/or signal transduction.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical . residues in non-critical regions of the polypeptide.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants of the human G2RF polypeptide that do not have the ability to modulate cytotoxin and/or metabolite levels (e.g., oxaloacetate, hydroxycarboxylic acid, thioester compound, or glutathione-conjugated compound levels).
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion or deletion in critical residues or critical regions.
  • the present invention further provides non-human orthologues (e.g., non-human orthologues of the human G2RF polypeptide).
  • Orthologues of the human G2RF polypeptides are polypeptides that are isolated from non-human organisms and possess the same ability to regulate cytotoxin and metabolite levels as the human G2RF polypeptide.
  • Orthologues of the human G2RF polypeptide can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2.
  • nucleic acid molecules encoding other G2RF family members and, thus, which have a nucleotide sequence which differs from the G2RF sequences of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number are intended to be within the scope of the invention.
  • another G2RF cDNA can be identified based on the nucleotide sequence of human G2RF.
  • nucleic acid molecules encoding G2RF polypeptides from different species and which, thus, have a nucleotide sequence which differs from the G2RF sequences of SEQ ID NO: 1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number are intended to be within the scope of the invention.
  • a mouse G2RF cDNA can be identified based on the nucleotide sequence of a human G2RF.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the G2RF cDNAs of the invention can be isolated based on their homology to the G2RF nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the G2RF cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the G2RF gene.
  • Orthologues, homologues and allelic variants can be identified using methods known in the art (e.g., by hybridization to an isolated nucleic acid molecule of the present invention, for example, under stringent hybridization conditions).
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • the nucleic acid is at least 50, 100,
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90%) identical to each other remain hybridized to each other.
  • stringent hybridization conditions include hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50%> formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C.
  • SSC sodium chloride/sodium citrate
  • a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
  • a preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45° C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42-50°C are also intended to be encompassed by the present invention.
  • SSPE lxSSPE is 0.15M NaCl, lOmM NaH 2 PO 4 , and 1.25mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15mM sodium citrate
  • additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.
  • blocking agents e.g., BSA or salmon or herring sperm carrier DNA
  • detergents e.g., SDS
  • chelating agents e.g., EDTA
  • Ficoll e.g., Ficoll, PVP and the like.
  • an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH 2 PO 4 , 7% SDS at about 65°C, followed by one or more washes at 0.02M NaH 2 PO 4 , 1% SDS at 65°C, see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81 :1991-1995, (or, alternatively, 0.2X SSC, 1% SDS).
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or 3 and corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural polypeptide).
  • allelic variants of the G2RF sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO: 1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , thereby leading to changes in the amino acid sequence of the encoded G2RF polypeptides, without altering the functional ability of the G2RF polypeptides.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of G2RF (e.g. , the sequence of SEQ ID NO:2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the G2RF polypeptides of the present invention e.g., those present in a metallo- beta-lactamase superfamily domain, are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the G2RF polypeptides of the present invention and other members of the G2RF family are not likely to be amenable to alteration.
  • nucleic acid molecules encoding G2RF polypeptides that contain changes in amino acid residues that are not essential for activity.
  • G2RF polypeptides differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least about 50%, 55%), 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2 (e.g., to the entire length of SEQ ID NO:2).
  • An isolated nucleic acid molecule encoding a G2RF polypeptide identical to the polypeptide of SEQ ID NO:2, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • amino acid substitutions such that one or more amino acid substitutions, additions or deletions are introduced into the encoded polypeptide. Mutations can be introduced into SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a G2RF polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a G2RF coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for G2RF biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined.
  • a mutant G2RF polypeptide can be assayed for the ability to: (1) interact with cytotoxins and metabolites (e.g., lactoylglutathione, a glutathione-conjugated metabolite, a hydroxycarboxylic acid, and the like), (2) catalyze the metabolism of a cytotoxin or metabolite (e.g., lactoylglutathione, a glutathione- conjugated metabolite, a hydroxycarboxylic acid, and the like), (3) hydrolyze of a thioester compound (e.g., lactoylglutathione, and the like), (4) catalyze the formation of a thioester conjugation on a substrate (e.g.
  • cytotoxins and metabolites e.g., lactoylglutathione, a glutathione-conjugated metabolite, a hydroxycarboxylic acid, and the like
  • cytotoxins and/or metabolites e.g., detoxify
  • maintain equilibrium of cytotoxins and/or metabolites (8) modulate cancer or tumor progression, (9) modulate cellular proliferation, (10) modulate tissue development (e.g., embryogenesis), (11) modulate differentiation, (12) modulate apoptosis, or (13) modulate energy metabolism.
  • nucleic acid molecules encoding G2RF polypeptides described above another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto.
  • the invention provides an isolated nucleic acid molecule which is antisense to a G2RF nucleic acid molecule (e.g., is antisense to the coding strand of a G2RF nucleic acid molecule).
  • An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire G2RF coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding G2RF.
  • the term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human G2RF corresponds to SEQ ID NO:3).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding G2RF.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding G2RF disclosed herein (e.g., SEQ ID
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of G2RF mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of G2RF mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of G2RF mRNA (e.g., between the -10 and +10 regions of the start site of a gene nucleotide sequence).
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothiG2RFe derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a G2RF polypeptide to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g. , by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1981) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
  • a ribozyme having specificity for a G2RF-encoding nucleic acid can be designed based upon the nucleotide sequence of a G2RF cDNA disclosed herein (i. e. , SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ).
  • a derivative of a G2RF cDNA disclosed herein (i. e. , SEQ ID NO:l or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ).
  • Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a G2RF- encoding mRNA.
  • G2RF mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418..
  • G2RF gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the G2RF (e.g. , the G2RF promoter and/or enhancers) to form triple helical structures that prevent transcription of the G2RF gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the G2RF e.g. , the G2RF promoter and/or enhancers
  • G2RF promoter and/or enhancers e.g., the G2RF promoter and/or enhancers
  • the G2RF nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of G2RF nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of G2RF nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al.
  • PNAs of G2RF can be modified, (e.g. , to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of G2RF nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g. , RNase H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and" specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a step wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P. J. et al. (1996) supra).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et ⁇ /. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1981) Proc. Natl. Acad. Sci. USA 84:648- 652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et ⁇ /. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1981) Proc.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • an endogenous G2RF gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous G2RF gene.
  • a heterologous DNA regulatory element for example, an endogenous G2RF gene which is normally "transcriptionally silent", i.e., a G2RF gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • a transcriptionally silent, endogenous G2RF gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous G2RF gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.
  • G2RF or recombinant polypeptides can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • G2RF polypeptides are produced by recombinant DNA techniques.
  • a G2RF polypeptide or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the G2RF polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of G2RF polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of G2RF polypeptide having less than about 30% (by dry weight) of non-G2RF polypeptide (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-G2RF polypeptide, still more preferably less than about 10% of non-G2RF polypeptide, and most preferably less than about 5% non-G2RF polypeptide.
  • non-G2RF polypeptide also referred to herein as a "contaminating protein”
  • the G2RF polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of G2RF polypeptide in which the polypeptide is separated from chemical precursors or other chemicals which are involved in the synthesis of the polypeptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of G2RF polypeptide having less than about 30% (by dry weight) of chemical precursors or non-G2RF chemicals, more preferably less than about 20% chemical precursors or non-G2RF chemicals, still more preferably less than about 10% chemical precursors or non-G2RF chemicals, and most preferably less than about 5% chemical precursors or non-G2RF chemicals.
  • a "biologically active portion" of a G2RF polypeptide includes a fragment of a G2RF polypeptide which participates in an interaction between a G2RF molecule and a non-G2RF molecule.
  • Biologically active portions of a G2RF polypeptide include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the G2RF polypeptide, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length G2RF polypeptides, and exhibit at least one activity of a G2RF polypeptide.
  • biologically active portions comprise a domain or motif with at least one activity of the G2RF polypeptide, e.g., modulating cytotoxin and/or metabolite levels.
  • a biologically active portion of a G2RF polypeptide can be a polypeptide which is, for example, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 or more amino acids in length.
  • Biologically active portions of a G2RF polypeptide can be used as targets for developing agents which modulate a G2RF mediated activity, e.g., modulating cytotoxin and/or metabolite levels.
  • a biologically active portion of a G2RF polypeptide comprises at least metallo-beta-lactamase superfamily domain. It is to be understood that a preferred biologically active portion of a G2RF polypeptide of the present invention comprises at least one or more of the following domains: a transmembrane domain and/or a metallo-beta-lactamase superfamily domain. Moreover, other biologically active portions, in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native G2RF polypeptide.
  • a fragment comprises at least 5 amino acids (e.g., contiguous or consecutive amino acids) of the amino acid sequence of SEQ ID NO:2, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as
  • a fragment comprises at least 10,
  • amino acids e.g., contiguous or consecutive amino acids
  • amino acid sequence of SEQ ID NO:2 amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number .
  • a G2RF polypeptide has an amino acid sequence shown in SEQ ID NO:2.
  • the G2RF polypeptide is substantially identical to SEQ ID NO:2, and retains the functional activity of the polypeptide of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the G2RF polypeptide is a polypeptide which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.
  • the invention features a G2RF polypeptide which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3, or a complement thereof.
  • This invention further features a G2RF polypeptide which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3, or a complement thereof.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%), even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the G2RF amino acid sequence of SEQ ID NO:2 having 282 amino acid residues, at least 85, preferably at least 113, more preferably at least 142, more preferably at least 170, even more preferably at least 198, and even more preferably at least 226 or 255 or more amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • GAP program in the GCG software package (available at http://www.gcg.com)
  • NWSgapdna.CMP matrix a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • parameters to be used in conjunction with the GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or version 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and polypeptide sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g. , XBLAST and NBLAST
  • G2RF chimeric or fusion proteins As used herein, a G2RF "chimeric protein” or “fusion protein” comprises a G2RF polypeptide operatively linked to a non-G2RF polypeptide.
  • An "G2RF polypeptide” refers to a polypeptide having an amino acid sequence corresponding to G2RF
  • a non-G2RF polypeptide refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially homologous to the G2RF polypeptide, e.g., a polypeptide which is different from the G2RF polypeptide and which is derived from the same or a different organism.
  • a G2RF fusion protein can correspond to all or a portion of a G2RF polypeptide.
  • a G2RF fusion protein comprises at least one biologically active portion of a G2RF polypeptide.
  • a G2RF fusion protein comprises at least two biologically active portions of a G2RF polypeptide.
  • the term "operatively linked" is intended to indicate that the G2RF polypeptide and the non- G2RF polypeptide are fused in-frame to each other.
  • the non-G2RF polypeptide can be fused to the N-terminus or C-terminus of the G2RF polypeptide.
  • the fusion protein is a GST-G2RF fusion protein in which the G2RF sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant G2RF.
  • the fusion protein is a G2RF polypeptide containing a heterologous signal sequence at its N-terminus.
  • G2RF polypeptide containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of G2RF can be increased through the use of a heterologous signal sequence.
  • the G2RF fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the G2RF fusion proteins can be used to affect the bioavailability of a G2RF substrate.
  • Use of G2RF fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a G2RF polypeptide; (ii) mis-regulation of the G2RF gene; and (iii) aberrant post- translational modification of a G2RF polypeptide.
  • the G2RF-fusion proteins of the invention can be used as immunogens to produce anti-G2RF antibodies in a subject, to purify G2RF ligands and in screening assays to identify molecules which inhibit the interaction of G2RF with a G2RF substrate.
  • a G2RF chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a G2RF- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the G2RF polypeptide.
  • the present invention also pertains to variants of the G2RF polypeptides which function as either G2RF agonists (mimetics) or as G2RF antagonists.
  • Variants of the G2RF polypeptides can be generated by mutagenesis, e.g. , discrete point mutation or truncation of a G2RF polypeptide.
  • An agonist of the G2RF polypeptides can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a G2RF polypeptide.
  • An antagonist of a G2RF polypeptide can inhibit one or more of the activities of the naturally occurring form of the G2RF polypeptide by, for example, competitively modulating a G2RF-mediated activity of a G2RF polypeptide.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the polypeptide has fewer side effects in a subject relative to treatment with the naturally occurring form of the G2RF polypeptide.
  • variants of a G2RF polypeptide which function as either G2RF agonists (mimetics) or as G2RF antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a G2RF polypeptide for G2RF polypeptide agonist or antagonist activity.
  • a variegated library of G2RF variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of G2RF variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential G2RF sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. , for phage display) containing the set of G2RF sequences therein.
  • fusion proteins e.g. , for phage display
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential G2RF sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et ⁇ Z. (1984) Annu. Rev. Biochem. 53:323; Itakura et ⁇ /. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.
  • libraries of fragments of a G2RF polypeptide coding sequence can be used to generate a variegated population of G2RF fragments for screening and subsequent selection of variants of a G2RF polypeptide.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a G2RF coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the G2RF polypeptide.
  • Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of G2RF polypeptides.
  • the most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
  • Recursive ensemble mutagenesis (REM) a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify G2RF variants (Arkin and Yourvan (1992) roc. Natl. Acad. Sci. USA 89:1811-1815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
  • cell based assays can be exploited to analyze a variegated G2RF library.
  • a library of expression vectors can be transfected into a cell line, e.g. , an endothelial cell line, which ordinarily responds to G2RF in a particular G2RF substrate-dependent manner.
  • the transfected cells are then contacted with G2RF and the effect of expression of the mutant on signaling by the G2RF substrate can be detected, e.g., by monitoring cytotoxin and/or metabolite (e.g., oxaloaldehydes, hydroxycarboxylic) concentrations.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the G2RF substrate, and the individual clones further characterized.
  • G2RF polypeptide can be used as an immunogen to generate antibodies that bind G2RF using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length G2RF polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments of G2RF for use as immunogens.
  • the antigenic peptide of G2RF comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of G2RF such that an antibody raised against the peptide forms a specific immune complex with G2RF.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of G2RF that are located on the surface of the polypeptide, e.g., hydrophilic regions, as well as regions with high antigenicity (see, for example, Figure 2).
  • a G2RF immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, gG2RF, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed G2RF polypeptide or a chemically synthesized G2RF polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic G2RF preparation induces a polyclonal anti-G2RF antibody response.
  • Another aspect of the invention pertains to anti-G2RF antibodies.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as G2RF.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind G2RF.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of G2RF.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular G2RF polypeptide with which it immunoreacts.
  • Polyclonal anti-G2RF antibodies can be prepared as described above by immunizing a suitable subject with a G2RF immunogen.
  • the anti-G2RF antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized G2RF.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against G2RF can be isolated from the mammal (e.g. , from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1916) Proc. Natl. Acad. Sci. USA 1 :2921 -31; zn ⁇ Y eh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds G2RF.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or S ⁇ 2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind G2RF, e.g., using a standard ELISA assay.
  • a monoclonal anti-G2RF antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display . library) with G2RF to thereby isolate immunoglobulin library members that bind G2RF.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791 ; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International
  • recombinant anti-G2RF antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US 86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No.
  • An anti-G2RF antibody (e.g., monoclonal antibody) can be used to isolate G2RF by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-G2RF antibody can facilitate the purification of natural G2RF from cells and of recombinantly produced G2RF expressed in host cells.
  • an anti-G2RF antibody can be used to detect G2RF polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the G2RF polypeptide.
  • Anti-G2RF antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling ( i. e. , physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • vectors for example recombinant expression vectors, containing a nucleic acid containing a G2RF nucleic acid molecule or vectors containing a nucleic acid molecule which encodes a G2RF polypeptide (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded D ⁇ A loop into which additional D ⁇ A segments can be ligated.
  • viral vector Another type of vector, wherein additional D ⁇ A segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to, which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in En ⁇ ymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g. , G2RF polypeptides, mutant forms of G2RF polypeptides, fusion proteins, and the like).
  • an exemplary embodiment provides a method for producing a polypeptide, preferably a G2RF polypeptide, by culturing in a suitable medium a host cell of the invention (e.g., a mammalian host cell such as a non-human mammalian cell) containing a recombinant expression vector, such that the polypeptide is produced.
  • a host cell of the invention e.g., a mammalian host cell such as a non-human mammalian cell
  • the recombinant expression vectors of the invention can be designed for expression of G2RF polypeptides in prokaryotic or eukaryotic cells.
  • G2RF polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.
  • telomeres Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • G2RF activity assays e.g., direct assays or competitive assays described in detail below
  • a G2RF fusion protein expressed in a retro viral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. , (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the G2RF expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al. , (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • G2RF polypeptides can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1981) EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.
  • promoters of T cell receptors Winoto and Baltimore (1989) EMBO J. 8:729-733 and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No.
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the - fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3 :537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to G2RF mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect of the invention pertains to host cells into which a G2RF nucleic acid molecule of the invention is introduced, e.g., a G2RF nucleic acid molecule within a vector (e.g., a recombinant expression vector) or a G2RF nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • a vector e.g., a recombinant expression vector
  • the terms "host cell” and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a G2RF polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Chinese hamster ovary cells
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g. , DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a G2RF polypeptide or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (/. e. , express) a G2RF polypeptide.
  • the invention further provides methods for producing a G2RF polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a G2RF polypeptide has been introduced) in a suitable medium such that a G2RF polypeptide is produced.
  • the method further comprises isolating a G2RF polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which G2RF-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous G2RF sequences have been introduced into their genome or homologous recombinant animals in which endogenous G2RF sequences have been altered.
  • Such animals are useful for studying the function and/or activity of a G2RF and for identifying and/or evaluating modulators of G2RF activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, gG2RFs, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous G2RF gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a G2RF- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the G2RF cDNA sequence of SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human G2RF gene such as a mouse or rat G2RF gene, can be used as a transgene.
  • G2RF gene homologue such as another G2RF family member, can be isolated based on hybridization to the G2RF cDNA sequences of SEQ ID NO: 1 or 3, or the DNA insert of the plasmid deposited with ATCC as
  • transgene Accession Number (described further in subsection I above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a G2RF transgene to direct expression of a G2RF polypeptide to particular cells.
  • transgenic founder animal can be identified based upon the presence of a G2RF transgene in its genome and/or expression of G2RF mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a G2RF polypeptide can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a G2RF gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the G2RF gene.
  • the G2RF gene can be a human gene (e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non- human homologue of a human G2RF gene (e.g. , a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:l).
  • a mouse G2RF gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous G2RF gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous G2RF gene is functionally disrupted (i. e. , no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous G2RF gene is mutated or otherwise altered but still encodes functional polypeptide (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous G2RF polypeptide).
  • the altered portion of the G2RF gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the G2RF gene to allow for homologous recombination to occur between the exogenous G2RF gene carried by the homologous recombination nucleic acid molecule and an endogenous G2RF gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking G2RF nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced G2RF gene has homologously recombined with the endogenous G2RF gene are selected (see e.g., Li, E. et ⁇ l.
  • the selected cells can then injected into a blastocyst of an animal (e.g. , a mouse) to form aggregation chimeras (see e.g. , Bradley, A. in
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system See, e.g., Lakso et al. (1992) Proc.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration typically comprise the nucleic acid molecule, polypeptide, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, cG2RFings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a cG2RFing such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a G2RF polypeptide, G2RF modulator or an anti-G2RF antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a fragment of a G2RF polypeptide, G2RF modulator or an anti-G2RF antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a polypeptide or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.
  • appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • an animal e.g., a human
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • a G2RF polypeptide of the invention has one or more of the following activities: (1) interactions with cytotoxins and metabolites (e.g., lactoylglutathione, a glutathione-conjugated metabolite, a hydroxycarboxylic acid, and the like), (2) catalyze the metabolism of a cytotoxin or metabolite (e.g., lactoylglutathione, a glutathione-conjugated metabolite, a hydroxycarboxylic acid, and the like), (3) hydrolyze a thioester compound (e.g., lactoylglutathione, and the like), (4) catalysis of the formation of a thioester conjugation on a substrate (e.g., lactate, a hydroxycarboxylic acid), (5) modulation of signal transduction in a cell, (6) modulation of levels of cytotoxins and/or metabolites (e.g.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express G2RF polypeptide (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect G2RF mRNA (e.g., in a biological sample) or a genetic alteration in a G2RF gene, and to modulate G2RF activity, as described further below.
  • the G2RF polypeptides can be used to treat disorders characterized by insufficient or excessive levels of production of a G2RF substrate (e.g., levels of cytotoxins and/or substrates) or production of G2RF inhibitors.
  • the G2RF polypeptides can be used to screen for naturally occurring G2RF substrates, to screen for drugs or compounds which modulate G2RF activity, as well as to treat disorders characterized by insufficient or excessive production of G2RF polypeptide or production of G2RF polypeptide forms which have decreased, aberrant or unwanted activity compared to G2RF wild type polypeptide (e.g., cytotoxin- and metabolite-associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders).
  • the anti-G2RF antibodies of the invention can be used to detect and isolate G2RF polypeptides, to regulate the bioavailability of G2RF polypeptides, and modul
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to G2RF polypeptides, have a stimulatory or inhibitory effect on, for example, G2RF expression or G2RF activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a G2RF substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to G2RF polypeptides, have a stimulatory or inhibitory effect on, for example, G2RF expression or G2RF activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a G2RF substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of a G2RF polypeptide or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a G2RF polypeptide or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a G2RF polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate G2RF activity is determined. Determining the ability of the test compound to modulate G2RF activity can be accomplished by monitoring, for example, cytotoxin and/or metabolite levels (e.g., oxaloacetate, hydroxycarboxylic acid, thioester compound, or glutathione- conjugated compound levels).
  • cytotoxin and/or metabolite levels e.g., oxaloacetate, hydroxycarboxylic acid, thioester compound, or glutathione- conjugated compound levels.
  • the cell for example, can be of mammalian origin, e.g., a heart, placenta, lung, liver, skeletal muscle, thymus, kidney, pancreas, testis, ovary, prostate, colon, or brain cell.
  • mammalian origin e.g., a heart, placenta, lung, liver, skeletal muscle, thymus, kidney, pancreas, testis, ovary, prostate, colon, or brain cell.
  • the ability of the test compound to modulate G2RF binding to a substrate or to bind to G2RF can also be determined. Determining the ability of the test compound to modulate G2RF binding to a substrate can be accomplished, for example, by coupling the G2RF substrate with a radioisotope or enzymatic label such that binding of the
  • G2RF substrate to G2RF can be determined by detecting the labeled G2RF substrate in a complex.
  • G2RF could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate G2RF binding to a G2RF substrate in a complex. Determining the ability of the test compound to bind G2RF can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to G2RF can be determined by detecting the labeled G2RF compound in a complex.
  • compounds e.g., G2RF substrates such as cytotoxins and/or metabolites
  • G2RF substrates such as cytotoxins and/or metabolites
  • compounds can be labeled with 125 1, 35 S, C, 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with G2RF without the labeling of either the compound or the G2RF. McConnell, H. M. et al. (1992) Science 251:1906- 1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light- addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing a G2RF target molecule (e.g., G2RF substrates such as a cytotoxin and/or a metabolite) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the G2RF target molecule. Determining the ability of the test compound to modulate the activity of a G2RF target molecule can be accomplished, for example, by determining the ability of the G2RF polypeptide to bind to or interact with the G2RF target molecule.
  • a G2RF target molecule e.g., G2RF substrates such as a cytotoxin and/or a metabolite
  • Determining the ability of the test compound to modulate the activity of a G2RF target molecule can be accomplished, for example, by determining the ability of the G2RF polypeptide to bind to or interact with the G2RF target molecule.
  • Determining the ability of the G2RF polypeptide, or a biologically active fragment thereof, to bind to or interact with a G2RF target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the G2RF polypeptide to bind to or interact with a G2RF target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target, detecting catalytic/enzymatic activity of the target using an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g, luciferase), or detecting a target-regulated cellular response.
  • a reporter gene comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g, luciferase
  • an assay of the present invention is a cell-free assay in which a G2RF polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the G2RF polypeptide or biologically active portion thereof is determined.
  • Preferred biologically active portions of the G2RF polypeptides to be used in assays of the present invention include fragments which participate in interactions with non-G2RF molecules, e.g., fragments with high surface probability scores (see, for example, Figure 2). Binding of the test compound to the G2RF polypeptide can be determined either directly or indirectly as described above.
  • the assay includes contacting the G2RF polypeptide or biologically active portion thereof with a known compound which binds G2RF to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a G2RF polypeptide, wherein determining the ability of the test compound to interact with a G2RF polypeptide comprises determining the ability of the test compound to preferentially bind to G2RF or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a G2RF polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the G2RF polypeptide or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a G2RF polypeptide can be accomplished, for example, by determining the ability of the G2RF polypeptide to bind to a G2RF target molecule by one of the methods described above for determining direct binding.
  • Determining the ability of the G2RF polypeptide to bind to a G2RF target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BI A). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et ⁇ Z. (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • determining the ability of the test compound to modulate the activity of a G2RF polypeptide can be accomplished by determining the ability of the G2RF polypeptide to further modulate the activity of a downstream effector of a G2RF target molecule.
  • the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting a G2RF polypeptide or biologically active portion thereof with a known compound which binds the G2RF polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the G2RF polypeptide, wherein determining the ability of the test compound to interact with the G2RF polypeptide comprises determining the ability of the G2RF polypeptide to preferentially bind to or modulate the activity of a G2RF target molecule.
  • binding of a test compound to a G2RF polypeptide, or interaction of a G2RF polypeptide with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase/ G2RF fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized micrometer plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or G2RF polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or micrometer plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized micrometer plates which are then combined with the test compound or the test compound and either the non-adsorbed target protein or G2RF polypeptide, and the mixture incubated under conditions
  • the complexes can be dissociated from the matrix, and the level of G2RF binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • a G2RF polypeptide or a G2RF target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated G2RF polypeptide or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-cG2RFed 96 well plates (Pierce Chemical).
  • antibodies reactive with G2RF polypeptide or target molecules but which do not interfere with binding of the G2RF polypeptide to its target molecule can be derivatized to the wells of the plate, and unbound target or G2RF polypeptide trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the G2RF polypeptide or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the G2RF polypeptide or target molecule.
  • modulators of G2RF expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of G2RF mRNA or polypeptide in the cell is determined.
  • the level of expression of G2RF mRNA or polypeptide in the presence of the candidate compound is compared to the level of expression of G2RF mRNA or polypeptide in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of G2RF expression based on this comparison. For example, when expression of G2RF mRNA or polypeptide is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of G2RF mRNA or polypeptide expression.
  • the candidate compound when expression of G2RF mRNA or polypeptide is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of G2RF mRNA or polypeptide expression.
  • the level of G2RF mRNA or polypeptide. expression in the cells can be determined by methods described herein for detecting G2RF mRNA or polypeptide.
  • the G2RF polypeptides can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g. , U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • G2RF-binding proteins proteins which bind to or interact with G2RF
  • G2RF-binding proteins proteins which bind to or interact with G2RF
  • G2RF-binding proteins are also likely to be involved in the propagation of signals by the G2RF polypeptides or G2RF targets as, for example, downstream elements of a G2RF -mediated signaling pathway.
  • G2RF -binding proteins are likely to be G2RF inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a G2RF polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the G2RF polypeptide.
  • a reporter gene e.g., LacZ
  • Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the G2RF polypeptide.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of a G2RF polypeptide can be confirmed in vivo, e.g. , in an animal such as an animal model for cellular transformation and/or tumorigenesis.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g. , a G2RF modulating agent, an antisense G2RF nucleic acid molecule, a G2RF-specific antibody, or a G2RF -binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the G2RF nucleotide sequences, described herein, can be used to map the location of the G2RF genes on a chromosome. The mapping of the G2RF sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • G2RF genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the G2RF nucleotide sequences. Computer analysis of the G2RF sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the G2RF sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the G2RF nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a G2RF sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre- screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the G2RF gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the G2RF sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the G2RF nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the G2RF nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • the noncoding sequences of SEQ ID NO:l can comfortably provide positive individual identification with a panel of perhaps 10 to 1 ,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from G2RF nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g. , hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO:l are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the G2RF nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:l having a length of at least 20 bases, preferably at least 30 bases.
  • the G2RF nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such G2RF probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., G2RF primers or probes can be used to screen tissue culture for contamination (i. e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the "field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • diagnostic assays for determining G2RF polypeptide and/or nucleic acid expression as well as G2RF activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted G2RF expression or activity.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with G2RF polypeptide, nucleic acid expression or activity. For example, mutations in a G2RF gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with G2RF polypeptide, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of G2RF in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of G2RF polypeptide or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting G2RF polypeptide or nucleic acid (e.g. , mRNA, or genomic DNA) that encodes G2RF polypeptide such that the presence of G2RF polypeptide or nucleic acid is detected in the biological sample.
  • the present invention provides a method for detecting the presence of G2RF activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of G2RF activity such that the presence of G2RF activity is detected in the biological sample.
  • a preferred agent for detecting G2RF mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to G2RF mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, the G2RF nucleic acid set forth in SEQ ID NO:l or 3, or the DNA insert of the plasmid deposited with ATCC as Accession
  • oligonucleotide of at least 15, 30, 50,
  • a preferred agent for detecting G2RF polypeptide is an antibody capable of binding to G2RF polypeptide, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect G2RF mRNA, polypeptide, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of G2RF mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of G2RF polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of G2RF genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of G2RF polypeptide include introducing into a subject a labeled anti- G2RF antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a G2RF polypeptide; (ii) aberrant expression of a gene encoding a G2RF polypeptide; (iii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a G2RF polypeptide, wherein a wild-type form of the gene encodes a polypeptide with a G2RF activity.
  • "Misexpression or aberrant expression” refers to a non-wild type pattern of gene expression, at the RNA or protein level.
  • Non-wild type levels e.g., over or under expression
  • a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage
  • a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus).
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting G2RF polypeptide, mRNA, or genomic DNA, such that the presence of G2RF polypeptide, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of G2RF polypeptide, mRNA or genomic DNA in the control sample with the presence of G2RF polypeptide, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of G2RF in a biological sample can comprise a labeled compound or agent capable of detecting G2RF polypeptide or mRNA in a biological sample; means for determining the amount of G2RF in the sample; and means for comparing the amount of G2RF in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect G2RF polypeptide or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted G2RF expression or activity.
  • aberrant includes a G2RF expression or activity which deviates from the wild type G2RF expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant G2RF expression or activity is intended to include the cases in which a mutation in the G2RF gene causes the G2RF gene to be under-expressed or over- expressed and situations in which such mutations result in a non-functional G2RF polypeptide or a polypeptide which does not function in a wild-type fashion, e.g., a polypeptide which does not interact with a G2RF substrate such as a cytotoxin and/or a metabolite (e.g., oxaloacetates, hydroxycarboxylic acids, thioester compounds, glutathione-conjugated compounds).
  • the term "unwanted” includes an unwanted phenomenon involved in a biological response, such as cellular proliferation.
  • unwanted includes a G2RF expression or activity which is undesirable in a subject.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in G2RF polypeptide activity or nucleic acid expression, such as cytotoxin- and metabolite-associated disorders (e.g., a cell permeabilization, cell necrosis or apoptosis, triggering of second messenger, cell proliferation, cell motility, or signal transduction disorder).
  • a disorder associated with a misregulation in G2RF polypeptide activity or nucleic acid expression such as cytotoxin- and metabolite-associated disorders (e.g., a cell permeabilization, cell necrosis or apoptosis, triggering of second messenger, cell proliferation, cell motility, or signal transduction disorder).
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in G2RF polypeptide activity or nucleic acid expression, such as a cytotoxin- and a metabolite-associated disorder (e.g., a cell permeabilization, cell necrosis or apoptosis, triggering of second messenger, cell proliferation, cell motility, or signal transduction disorder).
  • a cytotoxin- and a metabolite-associated disorder e.g., a cell permeabilization, cell necrosis or apoptosis, triggering of second messenger, cell proliferation, cell motility, or signal transduction disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted G2RF expression or activity in which a test sample is obtained from a subject and G2RF polypeptide or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of G2RF polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted G2RF expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted G2RF expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents for a cytotoxin- and metabolite-associated disorder e.g., glyoxalase-associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and or signal transduction disorders.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted G2RF expression or activity in which a test sample is obtained and G2RF polypeptide or nucleic acid expression or activity is detected (e.g., wherein the abundance of G2RF polypeptide or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted G2RF expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in a G2RF gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in G2RF polypeptide activity or nucleic acid expression, such as a cytotoxin- and metabolite-associated disorder (e.g., glyoxalase- associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders).
  • a cytotoxin- and metabolite-associated disorder e.g., glyoxalase- associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a G2RF -polypeptide, or the mis- expression of the G2RF gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a G2RF gene; 2) an addition of one or more nucleotides to a G2RF gene; 3) a substitution of one or more nucleotides of a G2RF gene, 4) a chromosomal rearrangement of a G2RF gene; 5) an alteration in the level of a messenger RNA transcript of a G2RF gene, 6) aberrant modification of a G2RF gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a G2RF gene, 8) a non-wild type level of a G2RF-polypeptide, 9) allelic loss of a G2RF gene, and 10) inappropriate post- translational modification of a G2RF-polypeptide.
  • a preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g. , Landegran et al. (1988) Science 241 : 1077- 1080; and Nakazawa et ⁇ /. (1994) Proc.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a G2RF gene under conditions such that hybridization and amplification of the G2RF-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P.M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a G2RF gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in G2RF can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 1: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753- 759).
  • genetic mutations in G2RF can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the G2RF gene and detect mutations by comparing the sequence of the sample G2RF with the corresponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 14:560) o ⁇ Sanger ((1977) Proc. Natl. Acad. Sci. USA 14:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the G2RF gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type G2RF sequence with potentially mutant RNA or DNA obtained from 'a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85 :4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in G2RF cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes proteins that recognize mismatched base pairs in double-stranded DNA
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a G2RF sequence e.g., a wild-type G2RF sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. PatentNo. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in G2RF genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control G2RF nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of , the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such • cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a G2RF gene.
  • any cell type or tissue in which G2RF is expressed may be utilized in the prognostic assays described herein.
  • G2RF polypeptide e.g., the modulation of membrane excitability
  • the effectiveness of an agent determined by a screening assay as described herein to increase G2RF gene expression, polypeptide levels, or upregulate G2RF activity can be monitored in clinical trials of subjects exhibiting decreased G2RF gene expression, polypeptide levels, or downregulated G2RF activity.
  • the effectiveness of an agent determined by a screening assay to decrease G2RF gene expression, polypeptide levels, or downregulate G2RF activity can be monitored in clinical trials of subjects exhibiting increased G2RF gene expression, polypeptide levels, or upregulated G2RF activity.
  • a G2RF gene and preferably, other genes that have been implicated in, for example, a G2RF-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including G2RF, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates G2RF activity can be identified.
  • an agent e.g., compound, drug or small molecule
  • G2RF activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of G2RF and other genes implicated in the G2RF-associated disorder, respectively.
  • the levels of gene expression can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of polypeptide produced, by one of the methods as described herein, or by measuring the levels of activity of G2RF or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a G2RF polypeptide, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the G2RF polypeptide, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the G2RF polypeptide, mRNA, or genomic DNA in the pre-administration sample with the G2RF polypeptide, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent
  • an agent
  • increased administration of the agent may be desirable to increase the expression or activity of G2RF to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of G2RF to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • G2RF expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted G2RF expression or activity (e.g., glyoxalase-associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders).
  • a disorder associated with aberrant or unwanted G2RF expression or activity e.g., glyoxalase-associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders.
  • treatment includes the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to a cell or tissue from a subject, who has a diseases or disorder, has a symptom of a disease or disorder, or is at risk of (or susceptible to) a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or disorder, the symptom of the disease or disorder, or the risk of (or susceptibility to) the disease or disorder.
  • a “therapeutic agent” includes, but is not limited to, small molecules, peptides, polypeptides, antibodies, ribozymes, and antisense oligonucleotides.
  • prophylactic and therapeutic methods of treatment such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drag (e.g., a patient's "drag response phenotype", or “drag response genotype”).
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the G2RF molecules of the present invention or G2RF modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted G2RF expression or activity, by administering to the subject a G2RF or an agent which modulates G2RF expression or at least one G2RF activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted G2RF expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the G2RF aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a G2RF, G2RF agonist or G2RF antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell capable of expressing G2RF with an agent that modulates one or more of the activities of G2RF polypeptide activity associated with the cell, such that G2RF activity in the cell is modulated.
  • An agent that modulates G2RF polypeptide activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring target molecule of a G2RF polypeptide (e.g., a G2RF substrate), a G2RF antibody, a G2RF agonist or antagonist, a peptidomimetic of a G2RF agonist or antagonist, or other small molecule.
  • the agent stimulates one or more G2RF activities. Examples of such stimulatory agents include active G2RF polypeptide and a nucleic acid molecule encoding G2RF that has been introduced into the cell.
  • the agent inhibits one or more G2RF activities.
  • inhibitory agents include antisense G2RF nucleic acid molecules, anti-G2RF antibodies, and G2RF inliibitors. These modulatory. methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • glycolase enzyme inhibitors and substrates and their contemplated use in cancer therapy are discussed in Lo and Thornalley (1993) Biochem. Soc. Trans. 2 159S, and Murthy et al. (1994) J. Med. Chem. 37:2161- 2166.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a G2RF polypeptide or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) G2RF expression or activity.
  • the method involves administering a G2RF polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted G2RF expression or activity.
  • Stimulation of G2RF activity is desirable in situations in which G2RF is abnormally downregulated and/or in which increased G2RF activity is likely to have a beneficial effect.
  • inhibition of G2RF activity is desirable in situations in which G2RF is abnormally upregulated and/or in which decreased G2RF activity is likely to have a beneficial effect.
  • G2RF molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on G2RF activity (e.g., G2RF gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) G2RF-associated disorders (e.g., cytotoxin- and metabolite-associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders) associated with aberrant or unwanted G2RF activity.
  • G2RF-associated disorders e.g., cytotoxin- and metabolite-associated disorders such as cytotoxin and/or metabolite imbalance, cell necrosis, apoptosis, cell proliferation, cell differentiation, and/or signal transduction disorders
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a G2RF molecule or G2RF modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a G2RF molecule or G2RF modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1991) Clin. Chem. 43(2):254- 266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drags anti-malarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related, markers (e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.)
  • markers e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase 11/111 drug trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNP single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response.
  • a gene that encodes a drugs target e.g., a G2RF polypeptide of the present invention
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drag response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drag action.
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a G2RF molecule or G2RF modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • the G2RF molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drag activity, or as markers of the pharmacogenomic profile of a subject.
  • the presence, absence and/or quantity of the G2RF molecules of the invention may be detected, and may be correlated with one or more biological states in vivo.
  • the G2RF molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states.
  • a "surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder.
  • Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g.
  • an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS).
  • surrogate markers include: Koomen et al.. (2000) J Mass. Spectrum. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.
  • the G2RF molecules of the invention are also useful as pharmacodynamic markers.
  • a "pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects.
  • the presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drag in a subject.
  • a pharmacodynamic marker may be indicative of the concentration of the drag in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drag may be monitored by the pharmacodynamic marker.
  • the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo.
  • Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drag is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a G2RF marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drag itself.
  • the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-G2RF antibodies may be employed in an immune-based detection system for a G2RF polypeptide marker, or G2RF-specific radiolabeled probes may be used to detect a G2RF mRNA marker.
  • a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al.. US 6,033,862; Hattis et al. (1991) En. Health Perspex. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56: S21- S24; and Nicolau (l 999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.
  • the G2RF molecules of the invention are also useful as pharmacogenomic markers.
  • a "pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35(12): 1650-1652).
  • the presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug.
  • a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected.
  • RNA e.g., G2RF polypeptide or G2RF RNA
  • a drag or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject.
  • the presence or absence of a specific sequence mutation in G2RF DNA may correlate G2RF drug response.
  • the use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.
  • G2RF sequence information refers to any nucleotide and/or amino acid sequence information particular to the G2RF molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like.
  • SNPs single nucleotide polymorphisms
  • information "related to" said G2RF sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantitative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like.
  • “electronic apparatus readable media” refers to any suitable medium for storing, holding, or containing data or information that can be read and accessed directly by an electronic apparatus.
  • Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact discs; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybrids of these categories such as magnetic/optical storage media.
  • the medium is adapted or configured for having recorded thereon G2RF sequence information of the present invention.
  • the term "electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information.
  • Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatuses; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
  • sequence information refers to a process for storing or encoding information on the electronic apparatus readable medium.
  • Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the G2RF sequence information.
  • a variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium.
  • the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms.
  • Any number of dataprocessor structuring formats may be employed in order to obtain or create a medium having recorded thereon the G2RF sequence information.
  • G2RF sequence information in readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
  • the present invention therefore provides a medium for holding instructions for ⁇ performing a method for determining whether a subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder, wherein the method comprises the steps of determining G2RF sequence information associated with the subject and based on the G2RF sequence information, determining whether the subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder, and/or recommending a particular treatment for the disease, disorder, or pre-disease condition.
  • the present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a G2RF associated disease or disorder or a pre-disposition to a disease associated with G2RF wherein the method comprises the steps of determining G2RF sequence information associated with the subject, and based on the G2RF sequence information, determining whether the subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.
  • the present invention also provides in a network, a method for determining whether a subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder associated with G2RF, said method comprising the steps of receiving G2RF sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to G2RF and/or a G2RF associated disease or disorder, and based on one or more of the phenotypic information, the G2RF information (e.g. , sequence information and/or information related thereto), and the acquired information, determining whether the subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder.
  • a method for determining whether a subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder comprising the steps of receiving G2RF sequence information from the subject and/or information related thereto,
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the present invention also provides a business method for determining whether a subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder, said method comprising the steps of receiving information related to G2RF (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to G2RF and/or related to a G2RF associated disease or disorder, and based on one or more of the phenotypic information, the G2RF information, and the acquired information, determining whether the subject has a G2RF associated disease or disorder or a pre-disposition to a G2RF associated disease or disorder.
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the invention also includes an array comprising a G2RF sequence of the present invention.
  • the array can be used to assay expression of one or more genes in the array.
  • the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be G2RF. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.
  • the invention allows the quantitation of gene expression.
  • tissue specificity but also the level of expression of a battery of genes in the tissue is ascertainable.
  • genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues.
  • one tissue can be perturbed and the effect on gene expression in a second tissue can be determined.
  • the effect of one cell type on another cell type in response to a biological stimulus can be determined.
  • Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression.
  • the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect.
  • undesirable biological effects can be determined at the molecular level.
  • the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
  • the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a G2RF associated disease or disorder, progression of G2RF associated disease or disorder, and processes, such a cellular transformation associated with the G2RF associated disease or disorder.
  • the array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of G2RF expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
  • the array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells.
  • This provides a battery of genes (e.g., including G2RF) that could serve as a molecular target for diagnosis or therapeutic intervention.
  • the invention is based, at least in part, on the discovery of a human gene encoding a novel polypeptide, referred to herein as human G2RF.
  • human G2RF novel polypeptide
  • the entire sequence of the human clone 50566 was determined and found to contain an open reading frame termed human "G2RF.”
  • the nucleotide sequence of the human G2RF gene is set forth in Figure 1 and in the Sequence Listing as SEQ ID NO:l.
  • the amino acid sequence of the human G2RF expression product is set forth in Figure 1 and in the Sequence Listing as SEQ ID NO: 2.
  • the G2RF polypeptide comprises about 282 amino acids.
  • the coding region (open reading frame) of SEQ ID NO:l is set forth as SEQ ID NO:3.
  • the amino acid sequence of human G2RF was analyzed using the program PSORT (http://www.psort.nibb.ac.jp) to predict the localization of the proteins within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence. The results of the analyses show that human G2RF may be localized to the cytoplasm, nucleus, mitochondria, or golgi.
  • This example describes the tissue distribution of human G2RF mRNA, as may be determined by in situ hybridization analysis using oligonucleotide probes based on the human G2RF sequence.
  • tissues e.g. tissues obtained from brain
  • various tissues are first frozen on dry ice.
  • Ten-micrometer-thick sections of the tissues are postfixed with 4% formaldehyde in DEPC treated IX phosphate-buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH 8.0).
  • sections are rinsed in DEPC 2X SSC (IX SSC is 0.15M NaCl plus 0.015M sodium citrate).
  • Tissue is then dehydrated through a series of ethanol washes, incubated in 100% chloroform for 5 minutes, and then rinsed in 100%) ethanol for 1 minute and 95%) ethanol for 1 minute and allowed to air dry.
  • Hybridizations are performed with 35s-radiolabeled (5 X 10 ⁇ cpm/ml) cRNA probes.
  • Probes are incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55°C. After hybridization, slides are washed with 2X SSC.
  • Sections are then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNE with lO ⁇ g of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides are then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour.
  • TNE a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA
  • Sections are then dehydrated rapidly through serial ethanol-0.3 M sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and exposed at 4°C for 7 days before being developed and counter stained.
  • human G2RF is expressed as a recombinant glutathione-S- transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • G2RF is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
  • Expression of the GST-G2RF fusion polypeptide in PEB199 is induced with IPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.
  • the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire G2RF polypeptide and an HA tag (Wilson et al. (1984) Cell 31:161) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant polypeptide under the control of the CMV promoter.
  • the human G2RF DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the G2RF coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the G2RF coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the G2RF gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 , SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. COS cells are subsequently transfected with the human G2RF-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E.
  • the culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS- PAGE.
  • detergents 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5.
  • DNA containing the human G2RF coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the G2RF polypeptide is detected by radiolabelling and immunoprecipitation using a G2RF-specific monoclonal antibody.
  • EXAMPLE 4 TISSUE DISTRIBUTION OF HUMAN G2RF BY TAQMAN
  • Tissue Expression Analysis of G2RF mRNA Using Taqman Analysis This example describes the tissue distribution of human G2RF mRNA in a variety of cells and tissues, as determined using the TaqManTM procedure.
  • the TaqmanTM procedure is a quantitative, reverse transcription PCR-based approach for detecting mRNA.
  • the RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq GoldTM DNA Polymerase to cleave a TaqManTM probe during PCR.
  • cDNA was generated from the samples of interest, e.g. , tumor samples and normal samples, cell lines and the like,and used as the starting material for PCR amplification.
  • a gene-specific oligonucleotide probe (complementary to the region being amplified) was included in the reaction (i.e., the TaqmanTM probe).
  • the TaqManTM probe includes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5' end of the probe (such as FAM (6- carboxyfluorescein), TET (6-carboxy-4,7,2',7'-tetrachlorofluorescein), JOE (6-carboxy- 4,5-dichlor -2,7-dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6- carboxy-N,N,N',N'-tetramethylrhodamine) at the 3' end of the probe.
  • a fluorescent reporter dye covalently linked to the 5' end of the probe
  • TET 6-carboxy-4,7,2',7'-tetrachlorofluorescein
  • JOE 6-carboxy
  • the fluorescent signal from the 5' dye is quenched.
  • the 5' to 3' nucleolytic activity of taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal.
  • the PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a way of quantitating the initial template concentration. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence.
  • the probe specifically anneals between the forward and reverse primer sites.
  • the 5 '-3' nucleolytic activity of the AmpliTaqTM Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target.
  • the probe fragments are then displaced from the target, and polymerization of the strand continues.
  • the 3' end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product.
  • Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as ⁇ microglobulin which has been labeled with a different fluor on the 5' end (typically JOE).
  • a primer/probe set was designed using Primer Express software and primary cDNA sequence information.
  • Total RNA was prepared from a series of tissues using an RNeasy kit from Qiagen
  • First strand cDNA was prepared from one ⁇ g total RNA using an oligo dT primer and Superscript II reverse transcriptase (GIBCO-BRL).
  • cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Mock cDNA synthesis in the absence of reverse transcriptase resulted in samples with no detectable PCR amplification of the control gene confirms efficient removal of genomic DNA contamination.

Abstract

L'invention concerne des molécules d'acide nucléique isolées, dénommées molécules d'acide nucléique G2RF, qui codent pour de nouvelles molécules de glycolase humaines. L'invention concerne également des molécules d'acide nucléique antisens, des vecteurs d'expression recombinants contenant des molécules d'acide nucléique G2RF, des cellules hôtes dans lesquelles ces vecteurs d'expression ont été introduits, et des animaux transgéniques dans lesquels un gène G2RF a été introduit ou disrupté. L'invention concerne en outre des polypeptides G2RF isolés, des polypeptides hybrides, des peptides antigéniques, et des anticorps anti-G2RF, ainsi que des méthodes diagnostiques dans lesquelles les compositions décrites sont utilisées.
EP01964528A 2000-08-31 2001-08-31 50566 : un nouveau facteur apparente a la glyoxalase ii humaine et utilisations de ce facteur Withdrawn EP1313861A2 (fr)

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